Cell wall-lysing complex enzymes and a process for the production thereof

ABSTRACT

Complex enzymes which can lyse the cell wall of a variety of microorganisms such as bacteria, fungi, yeast, Basidiomycetes and chlorella are produced and recovered from cultivation of Pellicularia sasakii or Pellicularia filamentosa.

This is a continuation of application Ser. No. 314,933, filed Dec. 14,1972, now U.S. Pat. No. 3,890,198.

BACKGROUND OF THE INVENTION

This invention relates to novel complex enzymes which lyse, that is,disintegrate and dissolve the cell wall of various microorganisms, andthis invention further relates to a process for the production of suchcomplex enzymes.

It is known that yeasts and Chlorella etc., are cultured on large scaleto utilise the cultured microorganisms in foodstuffs and feeds, and alsothat bacteria and fungi are cultured to use the cultured microorganismsfor the recovery of useful substances such as antibiotics, protein,nucleic acid, amino acids, vitamins and enzymes from the culture.However, the cell wall of yeasts and Chlorella are so strong that theirdigestibility is poor in the use as foodstuff and feed. Accordingly ithas been proposed to treat the yeasts and Chlorella with such an enzymewhich digests the cell wall of these microorganisms, in order to improvetheir digestibility. Moreover, it has been proposed to treat the cultureof bacteria or fungi with a cell wall-lysing enzyme in order tofacilitate the extraction of useful intracellular substances from thecultured microorganisms.

SUMMARY OF THE INVENTION

We, the present inventors, have made extensive research to seek for suchenzymes which are capable of lysing the cell and particularly the cellwall of various microorganisms. As a result, we have now found thatcomplex enzymes which are produced by known strains of the genusPellicularia exhibit a high ability to lyse, that is, dissolve the cellsof various microorganisms, and that these complex enzymes can beobtained by cultivating the known strains of the genus Pellicularia in aculture medium containing assimilable carbon sources and assimilablenitrogen sources to produce and accumulate in the medium, and thenisolating these complex enzymes from the medium.

According to a generic aspect of the present invention, therefore, thereis provided a process for the production of a cell wall-lysing complexenzyme, which comprises cultivating a known strain of the genusPellicularia in a culture medium containing assimilable carbon sourcesand assimilable nitrogen sources to produce and accumulate the complexenzyme in the medium, and then isolating the complex enzyme from themedium.

DETAILED DISCUSSION

According to the taxonomy, the genus Pellicularia falls within thefamily Corticiaceal of the order Aphyllophorales belonging to thesubclass Homolasidine of the class Basidomycetes. As known examples ofthe known strains of the genus Pellicularia which may be used accordingto the present invention for the production of the cell wall-lysingcomplex enzyme, there may be mentioned the known species Pelliculariasasakii which is known to cause a plant disease "sheath blight" in riceplant; and the known species Pellicularia filamentosa which is known tocause a plant disease "black scurf" in potato plant. There are knownsome strains of Pellicularia filamentosa deposited under ATCC Nos.14701, 14702, 14703 and 20206 and a strain of Pellicularia sasakiideposited under ATCC No. 13289 as shown on page 120 of the "Catalogue ofstrains" 9th Edition (in 1970) of the American Type Culture Collection.

We isolated a strain of Pellicularia sasakii from a lesion of the sheathblight infection in rice plant and deposited this strain under F.R.I.No. 1170 in a Japanese public depository "Fermentation ResearchInstitute", Agency of Industrial Science & Technology of Japan, Inage,Chiba City, Japan on 8th Nov., 1971.

This strain was deposited unrestricted with the American Type CultureCollection, Rockville, Md., on Dec. 12, 1972, and has been designatedATCC No. 20,365. We also isolated a strain of Pellicularis filamentosafrom a lesion of the black scurf infection in potato plant and depositedthis strain under F.R.I. No. 1171 on the same date. This strain wasdeposited unrestricted with the American Type Culture Collection,Rockville, Md. on Dec. 12, 1972, and has been designated ATCC No.20,366. Nevertheless, we are not able to differentiate the strainsF.R.I. Nos. 1170 and 1171 from the known strains of Pellicularia sasakiiand Pellicularia filamentosa, respectively, in view of the disclosure ina Japanese publication "Microbiology Handbook" pages 376-377 (publishedon 25th Dec., 1957 in Tokyo).

Morphological properties common to the abovementioned strains ofPellicularia sasakii and Pellicularia filamentosa are briefly describedbelow.

i. The vegetable mycelium is in fibrous form or pileus-shaped andinitially colorless and turning into yellowish brown color upon aging.

ii. The mycelium is generally of a diameter of 8-12 microns.

iii. The sclerotium is formed.

iv. The basidium is in the shape of barrel or obovoid and bears 4 or 8branches.

v. Colorless spores of smooth surface are produced.

vi. The hymenium is flat.

vii. The fruit body forms a reticulated membrane.

The species Pellicularia sasakii and Pellicularia filamentosa may bedifferentiated from each other in that Pellicularia sasakii is causativeof the sheath blight disease in rice plant; and Pellicularia filamentosais causative of the black scurf disease in potato plant and damping-offdisease in egg plant, and also in that P. sasakii forms blackish browncolored sclerotium of 5-13 mm, BASIDIUM MEASURING 18 × 9 microns andspore measuring 9 × 7 microns; and P. filamentosa forms brown coloredsclerotium of 1-3 mm, basidium measuring 15 × 8 microns and sporemeasuring 9 ×7 microns. However, some taxonomers have stated thatPellicularia sasakii is merely a natural variant of the speciesPellicularia filamentosa.

Any of the above-mentioned strains of P. sasakii and P. filamentosa maybe used according to the present invention to produce the cellwall-lysing complex enzyme. According to an embodiment of the presentinvention, therefore, there is provided a process for the production ofa cell wall-lysing complex enqyme, which comprises cultivating a knownstrain of Fellicularia sasakii or Pellicularia filamentosa in a culturemedium containing assimilable carbon sources and assimilable nitrogensources to produce and accumulate the complex enzyme in the medium, andthen isolating the enzyme complex from the medium.

In carrying out the process of the present invention, a strain of thegenus Pellicularia, particularly Pellicularia sasakii or Pelliculariafilamentosa may be cultivated either in a liguid culture medium or in asolid culture medium using known fermentation technique. It is generallysuitable to effect the cultivation of the Pellicularia strain at anincubation temperature of 15° - 40°C for a period of 48 - 240 hours,though the cultivation conditions may vary depending on the manner ofcultivation employed.

The culture medium in which the Pellicularia strain is cultivatedaccording to the process of the present invention should containassimilable carbon sources and nitrogen sources. Suitable examples ofthe carbon sources used are carbohydrates such as starch, glucose,sucrose, wheat bran, rice bran and the like. Suitable examples of thenitrogen sources are ammonium salts, nitrates, peptone, meat extract,yeast extract and defatted soybean meal, etc. It is feasible toincorporate a suitable amount of a growth-promoting substance such asvitamins into the culture medium employed. It is also possible to add apulver of dried yeast, Chlorella and/or mushroom etc., to promote theproduction of the enzymes. Furthermore, traces of inorganic salts suchas potassium salts, calcium salts, magnesium salts, iron salts,phosphates etc. may be incorporated into the culture medium to supplyvarious trace elements which are essential to the growth of thePellicularia strain.

The cultivation of the Pellicularia strain may proceed until asufficient amount of the complex enzyme desired have been produced andaccumulated in the culture medium. It is preferred to carry out thecultivation of the Pellicularia strain under aerobic conditions. Inliquid cultivation, it is preferable that the cultivation should beconducted at 20° - 35°C for a period of 48 - 120 hours under aerobic,submerged conditions. In solid cultivation, it is suitable that thecultivation is conducted at 20° - 35°C for 3 to 10 days.

The complex enzyme which is lytic to the cell of verious micooganisms isproduced and accumulated in the culture medium during the cultivation ofthe Pellicularia strain according to the present invention. A supernateof the culture broth which is obtained by filtering off or centrifugingthe solid matters such as mycelium and solid residue of the culturemedium therefrom may be used as such, for lysing the cell of variousmicroorganisms. Such a supernate may also be freeze-dried to give acrude enzyme preparation. When the solid cultivation has been conducted,the resulting solid culture may be extracted with water or bufferedwater of pH 4.0 - 7.0 to give an aqueous extract containing the complexenzyme, which may be utilized similarly to the above-mentioned filtrate.

In order to isolate the complex enzyme in the form of a more purifiedenzyme preparation, the culture medium containing the enzymes may befreed from the solid matter, for example, by filtration orcentrifugation, or it may be extracted with water or buffered water. Theresulting aqueous crude solution of the complex enzyme may then besubjected to a treatment with a known salting-out agent such as ammoniumsulfate, sodium chloide etc., or with a wate-miscible organic solventsuch as lower aliphatic alcohols, for example, methyl or ethyl alcoholor acetone to precipitate the complex enzyme from the crude solution.For further purification, the precipitated complex enzyme may bedissolved in an appropriate buffer solution such as 0.01 M acetatebuffered water and the enzyme solution may subsequently be subjected toa dialysis or gel-filtation process. The purified enzyme solution soobtained may then be freeze-dried to give the purified enzymepreparation essentially consisting of the desired complex enzyme.

The complex enzyme produced by Pellicularia sasakii and the complexenzyme produced by Pellicularia filamentosa according to the process ofthe present invention are soluble in water but insoluble in acetone andethanol, and they have the following common characteristics:

a. being active to lyse the living cells and dead cells of bacteria,fungi, yeasts, Basidiomycetes and Chlorella,

b. their cell wall-lysing activities being stable in a pH range of 3 -9, the optimum pH being in the range of 5 - 7,

c. being optimally active (cell wall-lysing activity) in a temperaturerange of 30° - 40°C, but the optimum temperature slightly varyingdepending on the nature of microorganisms of which cells are to belysed,

d. their cell wall-lysing activities being stable in a low temperaturerange but rapidly inactivated at a temperature of higher than 50°C,

e. their cell wall-lysing activities being inhibited by the presence ofMn⁺ ⁺, Ni⁺ ⁺ or Zn⁺ ⁺,

f. exhibiting the enzymatic activities of cellulase, glucanase,chitinase, protease, hemi-cellulase and carboxymethyl cellulase and

g. being essentially composed of the component enzymes each having themolecular weights of at least 50,000.

It is considered that the cell wall-lysing complex enzyme produced byPellicularia sasakii and the complex enzyme produced by Pelliculariafilamentosa are each a mixture of cellulase, glucanase, chitinase,protease, hemi-cellulase and carboxymethyl cellulase in the form of acomplex and exhibit their high activity of lysing the cell of variousmicroorganisms through the synergistic effect of the actions of theparticular component enzymes, though it is presumed that these complexenzymes produced by the different species of Pellicularia havecompositions which are more or less different from each other, as it isobserved that these complex enzymes show more or less differentproperties and potencies for the particular enzymatic activities.

According to a further aspect of the present invention, therefore, thereis provided a cell wall-lysing complex enzyme selected from the complexenzyme produced by a known strain of Pellicularia sasakii and thecomplex enzyme produced by a known strain of Pellicularia filamentosa,all these complex enzymes having the following common characteristics:

a. being active to lyse the living cells and dead cells of at leastAspergillus niger, Penicillium steckii, Saccharomyces cerevvisiae,Candida utilis, Candida albicans, Candida lipolitica, Lentinus edodes,Bacillus subtilis, Lactobacillus lactic, and Chlorella,

b. their cell wall-lysing activities being stable in a pH range of 3 to9, the optimum pH being in the range of 5 to 7,

c. their cell wall-lysing activities being optimal in a temperaturerange of 30° to 40°C, but the optimum temperature varying depending onthe nature of microorganisms of which cells are to be lysed,

d. their cell wall-lysing activities being stable in a low temperaturerange but rapidly inactivated at a temperature of higher than 50°C,

e. their cell wall-lysing activities being inhibited by the present ofMn⁺ ⁺, Ni⁺ ⁺ or Zn⁺ ⁺,

f. exhibiting the enzymatic activities of cellulase, carboxymethylcellulase, glucanase, chitinase, protease, hemi-cellulase and amylase,and

g. being essentially composed of the component enzymes each having themolecular weights of at least 50,000. The complex enzyme produced byPellicularia sasakii further exhibits such a cellulase activity whichhas an optimum temperature range of 40° to 50°C and an optimum pH of5.0, such a β-1,3-glucanase activity which has an optimum temperaturerange of 40° to 50°C an optimum pH of 5.0 and such a chitinase activitywhich has an optimum temperature of 35°C and an optimum pH range of 4.0to 5.5. The complex enzyme produced by Pellicularia filamentosa furtherexhibits such a cellulase activity which has an optimum temperaturerange of 40° to 50°C and an optimum pH of 5.0, such a β-1,3-glucanaseactivity which has an optimum temperature range of 40° to 50°C and anoptimum pH of 5.0, and such a chitinase activity which has an optimumtemperature of 35°C and an optimum pH range of 4.0 to 5.5.

The cell wall-lysing activities referred to in the above-mentionedcommon characteristics (b) - (e) of the complex enzymes of the presentinvention are determined by the following procedure and scale: An amountof a complex enzyme preparation obtained is reacted with 5 ml of asuspension of the cells of Candida utilis IFO-0396 which shows aninitial turbidity corresponding to an optical density (O.D.) value of1.0 as measured at a wave length of 660 milimicrons. The reaction iseffected at 35°C for 60 minutes at pH 5.0. After this reaction, theoptical density of the cell suspension containing the lysed cells andunlysed cells is measured at 660 milimicrons. When the reacted cellsuspension exhibits a reduction of 1% in the turbidity (or the O.D.value) as compared to the initial cell suspension, it is assumed thatsaid amount of the complex enzyme preparation has a potency of 1 unitfor the cell wall-lysing activity. Moreover, the cellulase activity,β-1,3-glucanase activity and chitinase activity referred to in the aboveof the complex enzymes of the present invention are measured in suchmanners as described later in Example 13 of this specification.

When the lysis, namely dissolution of the cell wall of a microorganismis effected using the complex enzyme of the present invention, thecomplex enzyme is reacted with the microorganism cells suspended inwater. The reaction may suitably be carried out at a temperature of20° - 60°C and preferably of 30° - 40°C at a pH of 2 - 10 and preferablyat a pH of 3.4 - 8.0. The reaction temperature and pH value employed maybe choosen appropriately depending on the nature of the microorganism ofwhich cells are to be lysed under the action of the complex ensyme. Themicroorganism cells to be lysed may either be living or dead when theyare brought into contact with the enzyme. In either case, substantiallyall the cells can be dissolved owing to the lysing of the cell wall andthe contents (intracellular substances) of the cells can be releasedinto the aqueous phase of the cell suspensions in 20 hours after thestart of the reaction.

In order to carry out the reaction of the complex enzyme with themicroorganism cells, either the enzyme preparation in the form of asolid powder or an aqueous solution thereof or the culture broth or anextract of the culture may be needed to a suspension of the cells inwater which has been buffered to a pH value optimal to effect theenzymatic reaction. Alternatively, the cells may be added to a solutionof the enzyme preparation in water or in a buffer solution which hasbeen adjusted to a pH value optimal to effect the enzymatic reaction.When the reaction has been effected to completion, the cells have whollybeen dissolved without leaving any solid residue in the solution. Insome cases, however, an amount of a solid residue remains even after thereaction was completed. Whether the cells have been dissolved to adesired degree, it can be estimated by determining the variation in theturbidity of the cell suspension or the concentration of a solublecomponent of the cell protoplasm, or by observing microscopically theconditions of the cells in the suspension. In this way, there may beobtained an aqueous cell solution in which the material of the cellmembranes and the protoplasm of the cells have been dissolved in watertogether with the complex enzyme employed. If desired, the cell solutionso obtained may be filtered to remove any possible solid residue. Thissolution may also be heated to deactivate the remaining enzymes, ifnecessary. The cell solution so obtained may subsequently be processedmerely be dehydration or concentration in vacuo to give a dry product.The cell solution may also be treated by a suitable means such asextraction or chromatography, to recover any useful substance orsubstances therefrom.

The complex enzyme of the present invention are able to lyse the cells(exactly speaking, the cell wall) of various microorganisms such asbacteria, fungi, yeast, Besidiomycetes as well as chlorella, one ofalgae. This property of the complex enzyme according to the presentinvention is more advantageous and useful, as compared to e.g. the knownenzyme which is isolated from the culture of Corticium rolfsii accordingto the method of Japanese patent publication No. 11978/67. The enzyme asisolated from the culture of Corticium rolfsii has an optimum pH rangeof 2.0 - 2.5 and a limited ability that they can lyse the cell wall ofyeast and chlorella only. The microorganisms of the genus Corticium canbe differentiated from those of the genus Pellicularia in that theformer produces such a continuous hymenium wherein the basidium arearranged side by side in a continuous manner, while the latter producessuch a discontinuous hymenium wherein the basidium are spaced from eachother.

According to an another aspect of the present invention, therefore,there is provided a method of dissolving the cells of microorganisms,including bacteria, fungi, yeasts, Basidiomycetes and chlorella, whichcomprises treating the cells suspended in an aqueous medium, with thecell wall-lysing complex enzyme which is by a known strain ofPellicularia sasakii or Pellicularia filamentosa or with a culture brothof said strain or an extract thereof.

The cell wall-lysing complex enzyme of the present invention may beapplied in the extraction of useful intracellular substances from thecells as well as in the production of a concentrated extract comprisingnutrient substances or other useful substances present in the protoplasmin the cells of various microorganisms such as yeasts, chlorella,bacteria and fungi. For these purposes the cells are treated with thecomplex enzyme in water to lyse the cell wall and to release theprotoplasm so as to give a cell solution which may then be dehydratedpartially or completely to give a concentrate or a dry powder.Furthermore, the cell wall-lysing complex enzyme of the presentinvention may be applied for the purpose of preventing foodstuff ordrinks from spoiling, by incorporating an effective amount of thecomplex enzyme therein and allowing to dissolve the cells of bacteria orfungi which would possibly occur or be present in the foodstuff ordrinks and to which the spoiling of the foodstuffs or drinks isattributable. Thus, the complex enzymes of the present invention findwide applications in many fields such as the food industry,pharmaceutical industry, pesticide industry and feed-preparing industry.

The present invention is now illustrated with reference to the followingExamples to which the invention is not limited in any way.

EXAMPLE 1

In a conical flask of a 300 ml. capacity were placed 19 g. of wheatbran, 1 g. of dried yeast and 18 ml. of water. The flask was then warmedand the content of the flask was agitated to give a uniform pastymixture. This culture medium was steamed at 120°C for 20 minutes forsterilization. A stock culture of Pellicularia sasakii (F.R.I. No. 1170)was inoculated to the sterile culture medium. Solid cultivation of thePellicularia strain was carried out for 120 hours at a constanttemperature of 28°C. The resulting culture was mixed with 80 ml. of anacetate buffer solution, pH 4.0, and the mixture was agitated at ambienttemperature for 1 hour to ensure that the enzyme were extracted into theliquid phase. The mixture was then filtered under pressure and thefiltrate obtained was centrifuged to give 70 ml. of a clear solutioncontaining the complex enzyme which was produced by Pelliculariasasakii. To this clear solution was added 210 ml. of acetone, so thatthe complex enzyme was precipitated. The precipitate was filtered outand dried to give 700 mg. of an enzyme preparation in the powder form.This enzyme preparation was found to be active to lyse the cell wall ofa variety of microorganisms, including bacteria, fungi, yeasts,Basidiomycetes and chlorella.

EXAMPLE 2

In a 300 ml. conical flask were placed 10 g. of wheat bran, 10 g. ofrice bran, 2 g. of peptone and 20 ml. of water. The flask was warmed andthe content of the flask was agitated to give a uniform pasty mixture.This culture medium was steamed at 120°C for 20 minutes forsterilization. A stock culture of Pellicularia filamentosa (F.R.I. No.1172) was inoculated to the sterile culture medium. Solid cultivation ofthe Pellicularia strain was carried out at 28°C for 170 hours. Theculture so obtained was then mixed with 80 ml. of water and the mixturewas well agitated at ambient temperature to ensure that the enzymes wereextracted into the aqueous phase. The mixture was filtered underpressure and the filtrate obtained was centrifuged to give a clearsolution containing the complex enzyme which was produced byPellicularia filamentosa. To the clear solution was added ammoniumsulfate to a 70% saturation, so that the complex enzyme wasprecipitated. The precipitate was filtered out and dried to give 1 g. ofa enzyme preparation in the powder form. This enzyme preparation wasfound to be active to lyse the cell wall of a variety of microorganisms,including bacteria, fungi, yeasts, Basidiomycetes and chlorella.

EXAMPLE 3

In an 1-liter conical flask were placed 9 g. of sucrose, 6 g. of ricebran, 0.6 g. of a powder of dried mushroom (Cortinellus shiitake), 1.5g. of ammonium sulfate, 1.5 g. of mono-potassium phosphate, 0.3 g. ofpotassium chloride, 0.3 g. of magnesium sulfate and 0.003 g. of ferroussulfate. To the content of the flask was added 300 ml. of water toeffect the dissolution of the soluble matter. The culture medium in theform of an aqueous solution was sterilized by steaming at 120°C for 20minutes. A stock culture of Pellicularia filamentosa (F.R.I. 1172) wasinoculated to the sterile liquid culture medium. Shake-cultivation ofthe Pellicularia strain was carried out at 25°C for 120 hours. Theculture broth so obtained was filtrated to remove the solid matterpresent, and the resulting clear filtrate containing the enzyme wasconcentrated to a volume of one-third the original volume, by dialysingthrough a dyalisis membrane. To the concentrated solution was added 400ml. of aqueous 99% ethyl alcohol, so that the complex enzyme wasprecipitated. The precipitate was filtered and dried to give 450 mg ofan enzyme preparation in the powder form. This enzyme preparation wasfound to be active to lyse the cell wall of a variety of microorganisms,including bacteria, fungi, yeasts, Basidiomycetes and chlorella.

EXAMPLE 4

In a 300 ml. conical flask were placed 5 g. of finely divided wheatbran, 0.5 g. of ammonium sulfate, 0.5 g. of mono-potassium phosphate,0.1 g. of potassium chloride, 0.1 g. of magnesium sulfate, 0.001 g. offerrous sulfate and 100 ml. of water. The content of the flask wassterilized by steaming at 120°C for 20 minutes. A stock culture ofPellicularia sasakii (F.R.I. No. 1170) was inoculated to the sterileliquid culture medium. Shake-incubation was carried out at 28°C for 24hours by means of a rotary shaker to prepare a liquid seed culture. Onthe other hand, 200 g. of wheat bran, 5 g. of a powder of dried mushroom(Cortinellus shiitake) and 160 ml. of an aqueous solution of 5% ammoniumsulfate were placed in 5 wooden trays, respectively. In each tray, thematerials were well mixed with each other to give a uniform mixturewhich was then sterilized by steaming. 20 ml. of the abovementionedliquid seed culture prepared was inoculated to the culture medium ineach tray. Stationary cultivation was conducted at 28° C for 96 hours.The cultures in these trays were combined together, and the combinedculture was extracted with 4 l. of an aqueous solution of acetate bufferpH 4.0. The extract was filtered under pressure and the filtrate wascentrifuged to give 3.6 l. of a clear solution containing the complexenzyme which was produced by Pellicularia sasakii. To this clearsolution was added ammonium sulfate to 70% saturation, so that thecomplex enzyme was precipitated. The precipitate was collected and takenup in 900 ml. of water. The aqueous enzyme solution was lyophillized invacuo to give 32 g. of an enzyme preparation in the form of a powder.This enzyme preparation was found to be active to lyse the cell wall ofa variety of microorganisms, including bacteria, fungi, yeasts,Basidiomycetes and chlorella.

EXAMPLE 5

In each of three conical flasks of a 300 ml. capacity were placed 19 g.of wheat bran, 1 g. of dried yeast and 18 ml. of water, and the contentsof each flask warmed and agitated to give a uniform pasty mixture. Thisculture medium was sterilized by steaming at 120°C for 20 minutes. Stockcultures of Pellicularia sasakii and Pellicularia filamentosa wereinoculated to the sterile culture media in these flasks, respectively.Solid cultivation was made for 120 hours at a constant temperature of28°C. After this cultivation, the cultures obtained were mixed with 80ml. portions of an acetate buffer solution and the mixtures wereagitated at ambient temperature for 1 hour to ensure that the complexenzymes produced by the different species of Pellicularia were extractedinto the liquid phases. The mixtures were filtered under pressure andthe filtrates obtained were centrifuged to give 70 ml. portions of twoclear solutions containing the complex enzymes, respectively.

On the other hand, cell suspensions of fungi, yeasts, bacteria andchlorella in water was prepared from cultures of Aspergillus niger,Penicillium steckii, Saccharomyces cerevisiae IFO-0209, Candida utilisIFO-0396, Candida albicans, Candida lipolitica and Lentinus edodes eachincubated in malt media; cultures of Bacillus subtilis and Lactobacilluslactis each incubated bouillon media; and culture of Chlorellapyrenoidosa incubated in Nakamura medium, respectively. Each of thesecell suspensions so prepared was placed in three test tubes. 20 ml.portions of the above-mentioned three clear solutions of the complexenzyme were then added to the three test tubes each containing the cellsuspensions, respectively. The content in each test tube was gentlyshaken for 20 hours in a water bath at a constant temperature of 35°C.,so that the enzymes were reacted with the cell wall to dissolve thecells in the suspension. The cell suspensions were observed undermicroscope to estimate how much is the degree to which the cells hadbeen dissolved or digested under the action of the complex enzymes ofPellicularia sasakii and Pellicularia filamentosa.

The degree of dissolution of the cells was estimated on the followingscale:

The symbol ++ denotes that there was observed the dissolution of all thecells.

The symbol + denotes that there was observed the dissolution of a largerpart of the cells.

The symbol ± denotes that there was observed the dissolution of a minorpart of the cells.

The symbol - denotes that there was not observed any dissolution of thecells.

The test results obtained are shown in Table 1 below. For comparison,the test procedure was repeated adding 2 ml. of the acetate buffersolution at instead of the above-mentioned clear enzyme solutions. Inthe comparative tests, no dissolution of the cells was observed.

                  TABLE 1                                                         ______________________________________                                                        Degree of dissolution                                                         of cells by                                                   Nature of the                                                                            Addition of                                                                              Enzyme of   Enzyme of                                   cells to be                                                                              enzyme     Pellicu-    Pellicu-                                    dissolved             laria       laria                                                             sasakii     filamentose                                 ______________________________________                                        Aspergillus                                                                              Added      ++          ++                                          niger      Not added  -           -                                           Penicillium                                                                              Added      ++          ++                                          steckii    Not added  -           -                                           Saccharomyces                                                                            Added      ++          ++                                          cerevisiae Not added  -           -                                           Candida    Added      ++          ++                                          utilis     Not added  -           -                                           Candida    Added      ++          ++                                          albicans   Not added  -           -                                           Candida    Added      ++          ++                                          lipolitica Not added  -           -                                           Lentinus   Added      +           +                                           edodes     Not added  -           -                                           Bacillus   Added      ±        ±                                        subtilis   Not added  -           -                                           Lactobacillus                                                                            Added      +           +                                           lactis     Not added  -           -                                           Chlorella  Added      +           +                                           pyrenoidosa                                                                              Not added  -           -                                           ______________________________________                                    

EXAMPLE 6

Cell suspensions in physiological saline water were prepared fromcultures of Candida utilis IFO-0396, Saccharomyces cerevisiae IFO-0209and Hansenula anomala IFO-0122 which were each obtained byshake-cultivating the microorganism in a liquid culture mediumcomprising 3% glucose, 0.8% (NH₄)₂ SO₄, 0.5% KH₂ PO₄, 0.2% NaCl, 0.2%MgSO₄.sup.. 7H₂ O and 0.1% yeast extract (by weight), pH 5.2, for aperiod of 20 hours while the shaking was made by means of a rotator (200r.p.m.). One drop of each of the cell suspensions so prepared was placedon a slide glass plate, and one drop of the clear solution of thecomplex enzyme of Pellicularia sasakii given in Example 1 was added tothe one drop of the cell suspension on the slide glass plate. Theliquids of these drops were rapidly mixed together and then covered witha covering glass plate. The slide glass plate was placed in a room at aconstant temperature of 30°C, and the change of the cells in the liquidmass on the glass plate was observed under microscope for a period oftime. It was observed that the cells were gradually dissolved as thetime lapsed. The degree of dissolution of the cells observed wasestimated on the following scale:

The symbol + denotes that the dissolution of the cells took place.

The symbol ++ denotes that the dissolution of the cells were advancing.

The symbol +++ denotes that the dissolution of all the cells had beencompleted.

The symbol - denotes that no change in the cells was observed.

The test results obtained are shown in Table 2 below. For comparison,the test procedure was repeated adding one drop of the acetate buffersolution, instead of the one drop of the enzyme solution. In thecomparative tests, no dissolution of the cells was observed.

                  TABLE 2                                                         ______________________________________                                                           Degree of dissolution of the                               Microorganism                                                                           Addition cells of the microorganisms                                to be     of       with lapse of time (minutes)                               dissolved enzyme   1     3   5   10   15  30  60  90                          ______________________________________                                        Candida   Added    +     ++  ++  +++                                          utilis    Not      -     -   -   -    -   -   -   -                                     added                                                               Saccharomyces                                                                           Added    -     -   +   +++                                          cerevisiae                                                                              Not      -     -   -   -    -   -   -   -                                     added                                                               Hansenula Added    -     -   -   -    +   ++  ++  +++                         anomala   Not      -     -   -   -    -   -   -   -                                     added                                                               ______________________________________                                    

EXAMPLE 7

In a 300 ml. conical flask were placed 20 g. of wheat bran, 0.5 g. of apowder of dried mushroom (Cortinellus shiitake) and 16 ml. of an aqueoussolution of 5% ammonium sulfate, and the contents of the flask warmedand agitated to give a uniform pasty mixture. This culture medium wassterilized by steaming at 120°C for 20 minutes. A stock culture ofPellicularia filamentosa was inoculated to the sterile culture medium,and solid cultivation thereof was conducted at 25°C for 144 hours. Theculture obtained was mixed with 80 ml. of de-ionized water and themixture was agitated at ambient temperature for 1 hour to ensure thatthe complex enzyme produced by Pellicularia filamentosa were extractedinto the liquid layer. The mixture was filtered under pressure and thefiltrate was centrifuged to give 65 ml. of a clear solution containingthe complex enzyme. To this clear solution was added ammonium sulfate to70% saturation, so that the complex enzyme was precipitated. Theprecipitate was filtered out and dried to yield 1.7 g. of an enzymepreparation in the form of a powder.

This enzyme preparation was dissolved in an acetate buffer solution, pH5.0 to prepare an enzyme solution container 1% of said enzymepreparation. 10 ml. of this enzyme solution was placed in a bent tube inthe shape of a letter L, and 1 g. of dried yeast (this yeast was aspecies of Candida which was incubated using a culture medium containingparaffinic hydrocarbon fraction of petroleum oil as the carbon sources)or 1 g. of dried chlorella was additionally placed into the L-shapedtube. The bent tube was shaken at a constant temperature of 40°C., sothat the enzymes were reacted with the cells of yeast or chlorella toeffect the lysis of the cell wall. After a reaction time of 1 hour or 3hours, 3 ml. of aqueous sodium hydroxide was added to the reactionmixture within the bent tube to adjust the pH to about 7. The mixturewas again shaken for further 15 minutes and then centrifuged to removethe solid matter therefrom. The clear solution obtained was analysed forthe contents of sugars and proteins which were dissolved out of thecells. The quantity of the sugars was determined by a colorimetricmethod at 530 mu using 3,5-dinitro-salicyclic acid as thecolor-developing agent and was expressed in the term of the quantity ofglucose. The quantity of the proteins was calculated by determining thenitrogen content in the proteins by the known Kjeldahl-ninhydrin method,and multiplying the determined nitrogen content by 6.25.

The test results obtained are shown in Table 3 below. For comparison,the test procedure was repeated adding 10 ml. of an acetate buffersolution, pH 5.0 in place of the above-mentioned enzyme solution.

                  TABLE 3                                                         ______________________________________                                                                       Contents of the                                                               substances                                                                    dissolved out                                  Microorganism                                                                            Addition  Reaction  of the cells                                   to be      of        time      (in mg.)                                       dissolved  enzyme    (in hour) Sugars Proteins                                ______________________________________                                        Dried      Added     1         70.2   291.0                                   yeast                3         75.4   368.2                                              Not added 1         5.7     95.1                                                        3         5.6     95.4                                   Dried      Added     1         30.3   162.2                                   chlorella            3         34.1   198.9                                              Not added 1         0.4     40.1                                                        3         0.4     40.6                                   ______________________________________                                    

EXAMPLE 8

This example is to show that the complex enzyme of the present inventionis useful to prevent various foodstuffs and drinks such as tomato juice,potato juice and orange juice from spoiling.

10 ml. Portions of tomato juice, potato juice or orange juice wereplaced in small dishes. On the other hand, the enzyme preparationproduced in Example 1 was purified by means of an ion-exchange resin.0.1 mg, 1 mg. and 10 mg. of the resulting purified enzyme preparationwere added to the contents of the dishes, respectively. Some dishescontaining the foodstuffs or drinks were not added with the enzymepreparation, as control. The dishes were maintained in air at 28°C for aperiod of time and then the occurrence of spoiling was checked byobserving discoloration, appearance and odor of the juices. The testresults are shown in Table 4 below. In this table, the symbol + denotesthe spoiling occurred and the symbol - denotes no spoiling occurred.

                  TABLE 4                                                         ______________________________________                                                            Time lapse                                                                    (days)                                                    Material   Addition of enzyme                                                                          1       2   4     7                                  ______________________________________                                        Tomato juice                                                                              Not added    -       +   +     +                                              0.1 mg/10 ml.                                                                              -       -   -     +                                              1 mg/10 ml.  -       -   -     -                                              10 mg/10 ml. -       -   -     -                                  Potato juice                                                                              Not added    +       +   +     +                                              0.1 mg/10 ml.                                                                              -       -   +     +                                              1 mg/10 ml.  -       -   +     +                                              10 mg/10 ml. -       -   -     -                                  Orange juice                                                                              Not added    -       -   +     +                                              0.1 mg/10 ml.                                                                              -       -   -     -                                              1 mg/10 ml.  -       -   -     -                                              10 mg/10 ml. -       -   -     -                                  ______________________________________                                    

EXAMPLE 9

To 100 g. of dried cell cake of a yeast Candida lipolitica was added 1liter of an aqueous solution (pH 5.0) of 0.5% of the enzyme preparationwhich was produced in Example 2. The mixture was kept at 35°C for 30minutes to effect the enzymatic reaction, and the reaction mixture wasthen added with 1 liter of aqueous 0.2 N sodium hydroxide. The mixturewas heated at 50°C for 30 minutes to ensure that the protein of theyeast cells was extracted into the aqueous phase. The aqueous extractobtained was centrifuged to remove the cell cake, and the supernatantsolution was adjusted to pH 4.0 by addition of 6N hydrochloric acid sothat the protein was precipitated at the isoelectric point. The quantityof protein precipitated and recovered was corresponds to 76% of thewhole protein content of the yeast cells. When the test procedure wasrepeated without the enzyme preparation, the quantity of the proteinprecipitated and recovered corresponded only to 15 % of the wholeprotein content of the yeast cells.

EXAMPLE 10

10 g. of a feed consisting of dried cells of Candida utilis was added to100 ml. of an aqueous solution (pH 6.0) containing 0.5% of the enzymepreparation which was produced in Example 3. The mixture was kept at35°C for 1 hour so that the enzymes reacted with the cell wall of theyeast. The reaction mixture was freeze-dried to give a powder feedconsisting of the enzyme-treated Candida yeast. The procedure wasrepeated without the enzyme preparation, to give a powder feedconsisting of the merely freeze-dried Candida yeast. The initial driedCandida yeast untreated, the enzyme-treated Candida yeast and the merelyfreeze-dried were treated with pepsine to estimate their digestibility.The results obtained are shown in Table 5 below.

                  TABLE 5                                                         ______________________________________                                        Samples             Digestibility by pepsin                                   ______________________________________                                        Dried Candida yeast untreated                                                                     58.4%                                                     (comparative)                                                                 Merely freeze-dried Candida                                                                       60.2%                                                     yeast (comparative)                                                           Candida yeast treated with                                                                        97.7%                                                     the complex enzyme of the                                                     present invention                                                             ______________________________________                                    

EXAMPLE 11

To a volume of a clear solution containing the complex enzyme ofPellicularia sasakii which was obtained in Example 5 was added atwo-fold volume of acetone, so that the complex enzyme was precipitated.The precipitate was collected and dried to give a powdery enzymepreparation. 20 g. of a wet cake (solid content; 48% by weight)consisting of the cells of Candida utilis was added to 100 ml. of anaqueous solution (pH 4.0) containing 1% of said powdery enzymepreparation. The mixture obtained was gently shaken at 35°C for 1 hourto effect the enzymatic reaction. After the reaction, the reactionmixture was centrifuged to remove the remaining solid matter. Thesupernatant solution was lyophillized to give 7.8 g. of a dry powderyextract of the soluble, intracellular substances of the cells of Candidautilis. By analysis, this dry powdery extract was found to contain 54.3%crude proteins, 4.0% crude fats, 1.7% crude fibrous materials, 6.4%ashes and 33.6% soluble nitrogenous substances.

When the process was repeated adding 100 ml. of an acetate buffersolution, pH 4.0 instead of the enzyme solution, there was obtained only1.7 g. of a dry powder after the lyophillisation of the supernatantsolution.

EXAMPLE 12

An inoculant of Candida albicans, a pathogenic yeast, was prepared byinoculating a slant culture of this yeast to 100 ml. of potato-glucosemedium, incubating at 30°C for 24 hours and then diluting the mediumwith water to a volume of 10 times the original volume thereof. One ml.portions of the inoculant so prepared (containing 6 × 10⁴ cell coloniesof yeast per ml.) were added to 10 ml. portions of a potato-glucosemedium (pH 6.0) containing 0.5, 1, 2, 4, 8, 16 and 32 mg/ml. of theenzyme preparation of Pellicularia sasakii of Example 1, respectively.The inoculated media were incubated stationarily at 35°C for 24 hours,and thereafter the number of the unlysed cells of Candida albicans wascounted. The results obtained are shown in Table 6 below.

                  TABLE 6                                                         ______________________________________                                                              Number of cell                                          Content of the enzyme preparation                                                                   colonies per ml. of                                     in the incubated media (mg./ml.)                                                                    the media                                               ______________________________________                                        None                  2 × 10.sup.6                                      0.5                   1 × 10.sup.5                                      1                     7 × 10.sup.4                                      2                     4 × 10.sup.4                                      4                     2 × 10.sup.2                                      8                     0                                                       16                    0                                                       32                    0                                                       ______________________________________                                    

EXAMPLE 13

Enzymatic activities were determined of the complex enzymes which wereproduced from the cultures of Pellicularia sasakii and Pelliculariafilamentosa by the processes of Examples 4 and 2, respectively. Thedetermination of the enzymatic activities were conducted in thefollowing manners:

i. Cellulase activity: 5 ml. of an enzyme solution in water was placedin an L-shaped tube, and two pieces of filter paper (measuring 1 cm by 1cm.) were immersed in the enzyme solution within the tube. The solutionwas shaken at 40°C and the time taken until the filter paper pieces hadbeen disintegrated completely was determined. Potency of the cellulaseactivity was calculated according to the following equation: ##EQU1##wherein (x) denotes the time (in minutes) taken until the filter paperpieces had been disintegrated completely, and (y) denotes the volume (inml.) of the enzyme solution employed.

ii. β-1,3-Glucanase activity: 0.5 ml. of a solution of 1% of laminarinwas placed in a test tube and then added with 0.5 ml. of a dilutedenzyme solution (pH 5.0) and the mixture was heated at 40°C for 30minutes. After this, the determination of the quantity of the glucosepresent in the reaction mixture was conducted colorimetrically. 1 mg. ofglucose formed in the reaction mixture was taken as corresponding to 1unit of the β-1,3-glucanase activity per 0.5 ml. of said diluted enzymesolution.

iii. Chitinase activity: This activity was measured using 1 ml. of asuspension of 1% of powdered (100 mesh) poly-N-acetylglucosamine, pH 5.0and reacting therewith 1 ml. of a diluted enzyme solution at 40°C for 2hours. Formation of 1 mcg./ml. of N-acetylglucosamine in the reactionmixture was taken as corresponding to 1 unit of the chitanase activityper ml. of said enzyme solution.

The optimal temperature and optimal pH for the respective enzymeactivities were also measured for the enzyme preparations produced byPellicularia sasakii and Pellicularia filamentosa. The results obtainedare shown in Tables 7 and 8 below.

                  TABLE 7                                                         ______________________________________                                        Properties of the complex enzyme                                              produced by P. sasakii                                                                                Optimal                                                              Potency  temperature                                                                              Optimal                                    Enzymatic activities                                                                         (u./ml)  (°C)                                                                              pH                                         ______________________________________                                        Cell wall-lysing                                                                             210      30 - 40    5 - 7                                      activity                                                                      Cellulase      500      40 - 50    5.0                                        β-1,3-Glucanase                                                                         17.0     40 - 50    5.0                                        Chitinase      12.5     35         4.0-5.5                                    ______________________________________                                    

                  TABLE 8                                                         ______________________________________                                        Properties of the complex enzyme                                              produced by P. filamentosa                                                                            Optimal                                                              Potency  temperature                                                                              Optimal                                    Enzymatic activities                                                                         (u./ml.) (°C)                                                                              pH                                         ______________________________________                                        Cell wall-lysing                                                                             220      30 - 40    5 - 7                                      Cellulase      500      40 - 50    5                                          β-1,3-Glucanase                                                                         17.2     40 - 50    5.0                                        Chitinase      12.0     35         4.0-5.5                                    ______________________________________                                    

What we claim is:
 1. A powdery complex enzyme isolated from Pelliculariaculture media, capable of lysing cell walls and having the followingcharacteristics:a. being active to lyse the cell walls of living cellsand dead cells of Aspergillus niger, Penicillium steckii, Saccaromycescerevisiae, Candida utilis, Canadida albicans, Candida lipolitica,Lentinus edodes, Bacillus subtilis, Lactobacillus lactis and Chlorella;b. having cell wall-lysing activities stable in a pH range of 3 to 9,the optimum pH being in the range of 5 to 7; c. said cell wall-lysingactivities being optimal in a temperature range of 30° or 40°C., but theoptimum temperature varying depending on the nature of themicroorganisms of which cells are to be lysed; d. said cell wall-lysingactivities being stable in a low temperature range but rapidlyinactivated at a temperature of higher than 50°C.; e. said cellwall-lysing activities being inhibited by the presence of Mn⁺ ⁺, Ni⁺ ⁺or Zn⁺ ^(+;) f. exhibiting the enzymatic activities of cellulase andβ-1,3-glucanase at an optimum temperature of 40°-50°C. and an optimum pHof 5.0, chitinase at an optimum temperature of about 35°C. and anoptimum pH of 4.0-5.5, protease, hemicellulase and amylase; and g.consisting essentially of component enzymes each having a molecularweight of at least 50,000.
 2. A complex enzyme according to claim 1,having a relative cell wall-lysing activity of about 210 units, arelative cellulase activity of about 500 units, a relativeβ-1,3-glucanase activity of about 17.0 units and a relative chitinaseactivity of about 12.5 units.
 3. A complex enzyme according to claim 1,obtained from Pellicularia sasakii ATCC 20,365.
 4. A complex enzymeaccording to claim 1, obtained from Pellicularia filamentosa.
 5. Aprocess for the production of a complex enzyme capable of lysing cellwalls of microorganisms, which comprises cultivating a strain of thegenus Pellicularia in a culture medium containing assimilable carbonsources and assimilable nitrogen sources to produce and accumulate thecomplex enzyme in the medium, and then isolating the complex enzyme fromthe medium.
 6. A process as claimed in claim 5 in which liquidcultivation of Pellicularia sasakii or Pellicularia filamentosa isconducted at a temperature of 20°-35°C. for a period of 48-120 hoursunder aerobic conditions.
 7. A process as claimed in claim 5 in whichsolid cultivation of Pellicularia sasakii or Pellicularia filamentosa isconducted at a temperature of 20°-35°C. for a period of 3 to 10 daysunder aerobic conditions.
 8. A process as claimed in claim 5, whichcomprises cultivating Pellicularia sasakii or Pellicularia filamentosain a culture medium containing bran as an assimilable nitrogen source toproduce and accumulate the complex enzyme in the medium, extracting themedium with water or buffered water to give an aqueous extract solutionof the complex enzyme, precipitating the complex enzyme from the extractand drying the resultant precipitate.
 9. A process as claimed in claim5, which comprises cultivating Pellicularia sasakii or Pelliculariafilamentosa in a liquid culture medium containing assimilable nitrogensources under aerobic conditions to produce and accumulate the complexenzyme in the liquid medium, filtering the resulting culture broth toremove solid matter, concentrating the clear filtrate obtained,precipitating the complex enzyme from the concentrated filtrate anddrying the resultant precipitate.
 10. A process according to claim 5, inwhich said Pellicularia strain is Pellicularia filamentosa.